As it is known, there are highly immunogenic antigens that are able to induce protective immune responses in a subject, whereas there are other antigens that do not induce said protective response or which induce a very weak immune response. Generally, the immune response of the host to a weakly immunogenic antigen may be stimulated by means of the joint administration of an adjuvant.
Adjuvants
An adjuvant is any substance that increases the immune response to an antigen with which it is mixed. Adjuvants mainly act by means of three mechanisms: i) forming an antigen or allergen deposit at the vaccine application site from which the biologically active product will be released during a variable period of time; ii) delivering the antigen or allergen to the antigen-presenting cells; and iii) inducing interleukin secretion.
Some classic examples of adjuvants are: aluminum salts (Alhydrogel) and catecholamines, (which enhance a Th2 response), and the lipopolysaccharide of gram-negative bacteria and certain CpG sequences (which enhance the Th1 response). On the other hand, numerous studies demonstrate that certain non-biological vectors such as microparticles (spherical particles of polymeric nature coating a substance) or liposomes (spherical vesicles with an aqueous central cavity coated by a variable number of bimolecular phospholipid and cholesterol films) can also act as adjuvants [Eldridge et al., Infect Immun, 59 (1991) 2978-2986; O'Hagan et al., Vaccine, 18 (2000) 1793-1801; Murillo et al., Vaccine, 30 (2001) 4099-4106].
Another type of non-biological vectors which can be considered for their use as adjuvants are the solid particle colloidal systems with less than a micrometer in size, also called nanoparticles, which are subdivided into matrix nanospheres and vesicular nanocapsules [Orecchioni and Irache, Formes pharmaceutiques pour application locale. Lavoisier Tech and Doc., Paris, (1996) 441-457]. Nanocapsules are vesicular systems formed by an inner cavity that is surrounded by a polymer membrane or wall. Nanospheres are matrix forms, formed by a three-dimensional polymer network. In both cases, the molecules of the biologically active substance can be dissolved, trapped or bound in the macromolecular structure (in the nanospheres) or encapsulated by the polymer membrane (in the nanocapsules), and it can even be adsorbed to the nanoparticle surface.
The distribution of nanoparticles in the organism is, in general, dependent on its physicochemical characteristics (mainly size and surface properties) determining its interaction with the biological medium. Therefore they are pharmaceutical forms that are particularly interesting as immunotherapy or vaccine adjuvants for the administration of antigens and/or allergens.
In general, the most important potentials provided by these vectors of non-biological origin are as follows [Couvreur & Puisieux. Adv. Drug Del. Rev., 10 (1993) 141-162]: (i) they protect the encapsulated material from chemical, enzymatic or immunological inactivation at the administration and action site; (ii) they improve the transport of the biologically active molecule to hard-to-reach locations and its penetration in the cell; (iii) they prolong the drug residence time in the organism and control its release; (iv) they increase action specificity by selective, effective and regular concentration of the encapsulated material in the cellular and/or molecular target; and (v) they increase the stability of the material that they incorporated during manufacture, transport and storage of the medicinal product.
Use of Adjuvants in Vaccination
The use of particulate adjuvants in the form of emulsions, microparticles, ISCOMS or liposomes has previously been evaluated by several investigation groups [review: Singh et al., Int J Parasitology 33 (2003) 469-478].
Antigen capture by “antigen-presenting cells” increases when these antigens are associated with polymeric particles or are included inside them. Biodegradable and biocompatible polyesters have been used in humans and animals for many years as controlled antigen release systems [Okada et al., J Pharm Sci, 12 (1995) 1-99; Putney et al., Nat Biotechnol, 16 (1998) 153-157]. Unlike aluminum adjuvants, microparticles are effective in inducing cellular and cytotoxic immune responses in mice [Nixon et al., Vaccine 14 (1996) 1523-1530; Maloy et al., Immunology 81 (1994) 661-667; Moore et al., Vaccine 13 (1995) 1741-1749]. Oral immunization with microparticles in mice induces potent immune responses at the mucosal and systemic levels compared to encapsulated antigens [Chalacombe et al., Immunology 176 (1992) 164-168; Eldridge et al., J Control Rel 11 (1990) 205-214; O'Hagan et al., Novel Delivery Systems for Oral Vaccines (1994) 175-205]. This ability is the result of its internalization by specialized cells of the mucosa-associated lymphoid tissue [O'Hagan, J Anat, 189 (1996) 477-482]. Mucosal immunization with different particulate systems has demonstrated its effectiveness against different pathogens, such as Bordetella pertussis [Chaill et al., Vaccine 13 (1995) 455-462; Jones et al., Vaccine 15 (1997) 814-817; Shahin et al., Infect Immun, 63 (1995) 1195-1200; Conway et al., Vaccine 19 (2001) 1940-1950], Chlamidia trachomatis [Whittum-Hudson et al., Nat Med 2 (1996) 1116-1121], Salmonella Typhimurium [Allaoui-Attarki et al., Infect Immun 65 (1997) 853-857] and Brucella [Murillo et al., Vaccine, 19 (2001) 4099-4106].
Use of Adjuvants in Immunotherapy
Allergic diseases are an emerging pathology caused by an adverse immune response (hypersensitivity reaction) to intrinsically innocuous macromolecules, called allergens. This hypersensitivity affects about 30% of the population worldwide, mainly in industrialized countries. It is the cause of diseases such as allergic rhinitis, extrinsic asthma, food allergies and allergies to drugs and insects [Settipane et al., Allergy Proc, (1994) 21-25].
In Spain, the prevalence of this type of diseases in the population of 4 to 17 years of age is 13.3%; among them, 6.4% appear as bronchial asthma, the death rate due to asthma in Spain being 1.5/100,000 inhabitants.
The mechanistic theory of the cause of allergic diseases argues that they occur due to an altered balance between the two fundamental types of responses that may be generated after activating the T helper cells: Th1 and Th2. Cytokines present in the medium outside the cell affect in a determining manner the differentiation of immature T cells (Th0), such that the presence of interleukin 12 (IL-12), interferon gamma (IFN-γ), interleukin 18 (IL-18) and interferon alpha (IFN-α), induce differentiation towards Th1, which will mainly be characterized by the production of large quantities of IFN-γ, and, to a lesser degree, of interleukin 2 (IL-2) and interferon beta (IFN-β). The subsequent stimulation of B cells in this type of response will give rise to the production of IgG2a, IgG2b and IgG3. On the other hand, if the Th0 cell is in an environment in which interleukin 4 (IL-4) and prostaglandin E2 (PGE2) are predominant, differentiation towards Th2 will be induced, being characterized by the synthesis of large quantities of IL-4, interleukin 5 (IL-5) and interleukin 13 (IL-13), and by the synthesis of IgG1 and IgE, a biotype directly involved in the triggering process [Hannah et al., Ann Rev Immunol, 21 (2003) 579-628].
The importance of the predominance of a Th2, allergenic-specific type response in allergic diseases has been corroborated by a large number of studies [Romagnani, Ann Rev Immunol, 12 (1994) 227; Bousquet et al., Allergy, 53 (1998) 1-42; Majori et al., Clin Exp Allergy, 30 (2000) 341-347]. It has been demonstrated both in animal models and in man, that cells with a Th2 phenotype are the only cells able to directly recognize allergenic peptides and participate in the production of IgE by B cells, mastocyte activation and production, maturation and activation of eosinophils [Cohn et al., Pharmacology and Therapeutics, 88 (2000) 187-196].
Therefore, the functional predominance of Th2 over Th1 cells would lead to the allergic response, whereas the functional predominance of Th1 over Th2 cells would inhibit it [Martin et al., Alergol Immunol Clin, 17 (2002) 104-110].
Other studies claim that inhibition of a Th2 response with Th1 predominance could lead to the development of autoimmune diseases, so it would be more correct to enhance an immune regulation of the Th1/Th2 balance by increasing the population of regulatory T cells (Tr) and IL-10, and T cell growth factor β (TGF-β). This would lead to the synthesis of IgG4 and IgA antibodies (not inflammatory response mediators), and to the suppression of IgE production by B cells [Akdis et al., Immunology, 103 (2001) 131-136; Akdis et al., J Clin Invest, 102 (1998) 98-106; Blaser et al., Int Arch Allergy Immunol, 117 (1998) 1-10]. Recent studies reaffirm the importance of IL-10 in Th2 cell inactivation (Grunig et al., J Exp Med, 185 (1997) 1089-1099; Adachi et al., Int Arch Allergy Immunol, 118 (1999) 391-394], and it has even been found that in vivo administration of IL-10 has beneficial consequences in allergic animals [Zuany-Amorim et al., J Clin Invest, 95 (1995) 2644-2651; Stampfli et al., Am J Respir Cell Mol Biol, 21 (1999) 586-596; Hall et al., Vaccine, 21 (2003) 549-561]. This allows supposing that IL-10 plays an important regulatory role in Th2 cell hyperreactivity characteristic in allergic patients.
IL-10 can have important physiopathological implications for counteracting inflammatory diseases (Crohn's disease, rheumatoid arthritis, psoriasis, etc.), certain viral infections (hepatitis C, human immunodeficiency virus (HIV)-induced infections, etc.), or even inhibiting organ transplant side effects. Therefore, the direct application of IL-10, or else the use of adjuvants stimulating the production of IL-10, could have a huge impact on the treatment of these diseases [Asadullah et al., Pharmacol Rev, 55, (2003) 241-269]. It is currently being studied as a possible treatment for autoimmune diseases such as rheumatoid arthritis [Feldman et al., Annu Rev Immunol (1996) 397-440; Katsikis et al., J Exp Med (1994) 1517-1527; Chomarat et al., J Immunol (1995) 1432-1439]. The antiinflammatory and regulatory role of the cytokine therefore makes it essential in both Th1 (autoimmune diseases) and Th2 (allergy) excessive responses.
The treatment of allergic diseases may essentially be approached in three different manners: (i) avoiding all contact with the allergen; (ii) using antihistaminic drugs, and (iii) by means of immunotherapy. Bearing in mind that the first two measures are occasionally not applicable, immunotherapy would be the most suitable control method.
Specific immunotherapy with allergens has been defined as the repeated administration of allergens to patients with IgE-mediated health disorders for the purpose of providing protection against allergic symptoms and inflammatory reactions associated with the natural exposure to these allergens [Jutel, M., J Immunol, 154 (1995) 4178-4194].
This treatment alternative is aimed at enhancing a functional predominance of the Th1 response with respect to the Th2 response, which will inhibit the allergic symptomatology. This modulation towards Th1 is also applicable in other processes such as control by means of vaccination against bacterial intracellular parasites (such as Brucella and Salmonella).
Although the use of different non-biological vectors, for example nanoparticles, as adjuvants in immunotherapy or in vaccines for the administration of antigens and/or allergens has been described, there is still a need to provide alternative adjuvants to those currently existing for the purpose of increasing the arsenal of possibilities for manufacturing vaccines and compositions for immunotherapy. Advantageously, said adjuvants must be useful for their use in immunization or immunotherapy by oral administration without the need to use very high allergen and antigen doses. As is known, despite its potential advantages, oral immunization for therapeutic or prophylactic purposes must confront several obstacles since the dose of the immunogenic or allergenic active ingredient required for a beneficial clinical effect is extremely high due to an immunogen potency loss. Therefore, generally due to the little stability of the allergen or antigen in the gastrointestinal tract (pH conditions and presence of hydrolytic enzymes), doses must always be much higher (up to 200 times higher) than those normally used subcutaneously [Taudorf et al., J Allergy Clin Immunol (1987) 153-161; Creticos et al., J Allergy Clin Immunol (1990) 165]. Furthermore, the gastrointestinal mucosa acts as a rather impermeable barrier against the absorption of these macromolecules.
Surprisingly, it has now been found that methyl vinyl ether-maleic anhydride copolymer-based nanoparticles, optionally containing an allergen or antigen and/or an immunostimulating agent, have the ability to stimulate or enhance immune response when they are administered to a subject, which allows their use in immunotherapy and vaccines. Said nanoparticles are stable in oral administration, have good bioadhesive characteristics, and can therefore be used in immunization or immunotherapy through different administration routes, including the oral route, without needing to use such high allergen or antigen doses as those mentioned in the state of the art. Furthermore, said nanoparticles are low toxic, they are biodegradable and easy to produce.
Methyl Vinyl Ether-Maleic Anhydride Copolymer Nanoparticles
Patent application WO 02/069938, belonging to the same applicant, discloses methyl vinyl ether-maleic anhydride (PVM/MA) copolymer nanoparticles, a process for obtaining them and their use as drug carriers. Said PVM/MA copolymer structurally consists of two differentiated functional groups having different solubility characteristics: a hydrophobic ester group and an anhydrous group. The carboxylic group is a solubilizing agent, since it tends to dissolve the polymer when it is ionized, and the ester group is hydrophobic, as it delays penetration of water into the polymer [Heller et al., J Appl Polym Sci, 22 (1978) 1991-2009]. Synthetic PVM/MA copolymers have very different applications. Gantrez® AN is widely used as a thickener and flocculating agent, dental adhesive, excipient in oral tablets, excipient in transdermal patches, etc. On the other hand, the use of these copolymers for controlled release of drugs has been disclosed [Heller et al., J Appl Polym Sci, 22 (1978) 1991-2009] and, in matrix forms, for topical release of drugs in the eye [Finne et al., J Pharm Sci, 80 (1991) 670-673; Finne et al., Int J Pharm, 78 (1992) 237-241].
PVM/MA-based nanoparticles have bioadhesive characteristics [Arbós et al., Int J Pharm, (2002) 129-136] so that when they are administered orally, they may interact with Peyer's patches, which contain 20% of all the lymphocytes of the organism, and trigger an amplified immune response with regard to that of antigens and/or allergens administered in aqueous solution.
PVM/MA-based nanoparticles are colloidal systems that are able to retain biologically active substances by means of: (i) solution or entrapping inside the macromolecular structure or matrix, (ii) covalent bond of the drug with the copolymer anhydride groups and (iii) adsorption processes mediated by weak bonds. The extreme reactivity of the PVM/MA copolymer, due to cyclic anhydride groups, also contributes to its ability to entrap molecules, drugs or other substances. Patent application WO 02/069938 discloses the use of said PVM/MA copolymer nanoparticles as carriers for drugs, specifically 5-fluouridine, ganciclovir and antisense oligonucleotide ISIS 2922.